Nonmuscle myosin is present in all eukaryotic cells. It appears to play an important role in processes such as cell motility, cytokinesis, secretion and receptor capping. At least two different isoforms of nonmuscle myosin have been identified in vertebrates, referred to as NMHC-A and NMHC-B. Each may perform different cellular tasks. Recent studies identified neuronal-specific isoforms of nonmuscle myosin IIB, NMHC-B1 and NMHC-B2, which contain inserts of 10 and 21 amino acids near the ATP-binding and actin-binding regions, respectively. mRNA expression of these two isoforms differs in different regions of the brain and also differs at different stages of development. NMHC-B1 is expressed very early in development, while NMHC-B2 is expressed only after birth, mostly in the adult cerebellum. Almost nothing is known about the function of these neuronal-specific isoforms. This project is designed to investigate the pathophysiological roles of neuronal- specific NMHC-B2 by ablating the gene in mice. A 9.1 kb genomic DNA containing the 63 nt B2 insert was cloned from a 129/Sv mouse genomic library. A targeting vector was constructed by removing a SpeI/StuI fragment which contains the B2 insert and replacing it with a 1.8 kb PGK-Neo cassette flanked by two LoxP sites in the opposite orientation to myosin. To enhance screening efficiency, a 2.8 DT-A transcriptional cassette was inserted at the 3 end of the subclone in the same orientation as PGK-Neo. The targeting vector was then linearized by NotI and introduced into embryonic stem cells via electroporation. The purpose of this study is to understand the function of this insert during mouse brain development as well as in the adult mouse. Therefore, the relevant characterization of mouse brain function is being planned. Recently, in collaboration with Victor Ferrans group, we have localized noninserted NMHC-B to the Z-line and intercalated disks of the heart. Since mice hypomorphic for NMHC-B develop cardiac myocyte hypertrophy, we have initiated a study looking for mutations in NMHC-B using human tissue. The study is being carried out with Neal Epstein (Cardiology Branch, NHLBI). - nonmuscle myosin II-B; brain alternative splicing